CircTRRAP Knockdown Has Cardioprotective Function in Cardiomyocytes via the Signal Regulation of miR-370-3p/PAWR Axis

Background Circular RNA Transformation/Transcription Domain Associated Protein (circTRRAP, hsa_circ_0081241) was abnormally upregulated in acute myocardial infarction (AMI) patients. However, its biological role and functional mechanism in AMI remain to be researched. Methods Human cardiomyocyte AC16 was exposed to hypoxia to induce cell injury. Cell viability was detected through Cell Counting Kit-8. CircTRRAP, microRNA-370-3p (miR-370-3p), and Pro-Apoptotic WT1 Regulator (PAWR) levels were assayed by reverse transcription-quantitative polymerase chain reaction. Cell proliferation analysis was performed via 5-ethynyl-2′-deoxyuridine (EdU) assay. Cell apoptosis was assessed using flow cytometry and caspase-3 activity assay. The protein levels were measured through western blot. Enzyme-linked immunosorbent assay was used to examine the release of inflammatory cytokines. Oxidative stress was assessed by the commercial kits. Dual-luciferase reporter assay, RNA immunoprecipitation, and RNA pull-down assays were performed for the validation of target interaction. Results CircTRRAP was highly expressed following hypoxia treatment in AC16 cells. Downregulation of circTRRAP promoted cell growth but inhibited apoptosis, inflammation, and oxidative stress in hypoxic cells. CircTRRAP could target miR-370-3p, and the regulatory effects of circTRRAP on the hypoxic cells were associated with the sponge function of miR-370-3p. PAWR served as the target for miR-370-3p, and it was regulated by circTRRAP/miR-370-3p axis. The protective role of miR-370-3p was achieved by downregulating the PAWR expression in hypoxia-treated AC16 cells. Conclusion These findings demonstrated that silence of circTRRAP exerted the protection against the hypoxia-induced damages in cardiomyocytes through regulating the miR-370-3p and PAWR levels.


Introduction
Acute myocardial infarction (AMI) is an emergency incident with the sudden and persistent loss of oxygen supply or increase of oxygen demand in the heart [1,2]. AMI leads to geometric, molecular, structural, and functional changes to evoke ventricular remodeling, and inflammatory response is triggered after myocardial injury [3]. Although the substantial improvement of prognosis has been achieved over the past decades, AMI is still an important cause of morbidity and mortality in human beings worldwide [4]. In addition, AMI has also become a potential threat to young individuals [5]. It remains crucial to explore novel biomarkers in the pathological process of AMI.
Noncoding RNAs with high specificity and sensitivity have been used as biological targets for clinical diagnosis and surveillance of AMI [6,7]. Circular RNAs (circRNAs) are regulators of genes by depending on the "sponge-like" function of microRNAs (miRNAs), then affecting the disease development in life processes [8]. Knockdown of cir-cROBO2 inhibited apoptosis of cardiomyocytes in AMI through decreasing the level of TRADD by the upregulation of miR-1184 [9]. Overexpression of circCDYL sponged 4793-5p to induce the APP downregulation, thus promoting the proliferation ability of myocardial cells [10]. Zhao et al. reported that circMACF1 suppressed the AMI progression via targeting the miR-500b-5p/EMP1 network [11]. Circular RNA Transformation/Transcription Domain Associated Protein (circTRRAP, hsa_circ_0081241) originates from TRRAP gene, and it was upregulated in AMI patients [12]. The exploration of function and mechanism for circTRRAP in AMI has never been performed.
Herein, we hypothesized that circTRRAP played a sponge role for miR-370-3p, and PAWR served as a target for miR-370-3p. Furthermore, whether PAWR level could be affected by circTRRAP via targeting miR-370-3p was analyzed. This study performed the research of function and mechanism of circTRRAP in hypoxia-induced cardiomyocyte injury in vitro.

Reverse Transcription-Quantitative Polymerase Chain
Reaction (RT-qPCR) Assay. AC16 cells were washed with phosphate buffer solution (PBS, Sigma-Aldrich), and RNA extraction by Total RNA Extractor (Sangon, Shanghai, China) was performed in accordance with the instruction book. ReverTra Ace® qPCR RT Kit (Toyobo, Kita-Ku, Osaka, Japan) was used for reverse transcription, and SYBR® Green Realtime PCR Master Mix (Toyobo) was applied for quantitative reaction. The miRNA quantification was performed using MystiCq® microRNA cDNA Synthesis Mix and MystiCq® microRNA® SYBR® Green qPCR ReadyMix™ (Sigma-Aldrich). Ct values were collected for relative level analysis via the 2 -ΔΔCt method. β-Actin has been used as the control gene for expression correction of circRNA and mRNA, while U6 was applied to standardize the miRNA level. The specific primers were synthesized by Sangon, and the sequences are exhibited in Table 1. 2.5. CircRNA Stability and Localization Assays. For stability detection, circTRRAP and linear TRRAP levels were assayed through RT-qPCR following treatment of RNase R (GENE-SEED, Guangzhou, China) for total RNA. For localization analysis, RT-qPCR detection for circTRRAP was conducted after isolation of nuclear and cytoplastic RNAs by PARIS™ Kit (Invitrogen). Glyceraldehyde-phosphate dehydrogenase (GAPDH) for cytoplasm and U6 for nucleus served as the control genes, respectively.
2.6. 5-Ethynyl-2 ′ -Deoxyuridine (EdU) Assay. 5 × 10 4 /well AC16 cells were plated onto the 96-well plates, followed by hypoxia treatment and cell transfection. The treated cells were performed with EdU staining as per the direction of EdU Cell Proliferation Kit (Sangon). The nuclei were stained with diamidine phenylindole (DAPI; Sangon), and cell analysis was performed through the fluorescence microscope (Olympus, Tokyo, Japan), followed by calculating the ratio of EdU-positive cells (EdU + DAPI) in total cells.  Cardiovascular Therapeutics USA). The apoptotic rate was indicated as the percentage of Annexin V+/PI-and Annexin V+/PI+ stained cells in total cells.
2.11. Oxidative Assay. Oxidative injury was analyzed by the detection of malondialdehyde (MDA), reactive oxygen species (ROS), and superoxide dismutase (SOD). MDA and ROS levels were examined according to the guidelines of MDA Assay Kit (Sigma-Aldrich) and Total Reactive Oxygen Species (ROS) Assay Kit (Invitrogen). SOD activity was determined through the SOD Assay Kit (Sigma-Aldrich) as per the user's manual.  3 Cardiovascular Therapeutics 2.13. RNA Immunoprecipitation (RIP) Assay. The binding between circTRRAP and miR-370-3p was determined through Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). The magnetic beads were enveloped with antibody against Argonaute-2 (Anti-AGO2) or immunoglobulin G (Anti-IgG), then incubated to cell lysates of AC16 at 4°C overnight. The input group (positive group) was set by lacking antibody incubation in beads. RNA complexes on the beads were isolated; then, RT-qPCR detection was performed for circTRRAP and miR-370-3p.
2.14. RNA Pull-Down Assay. Biotin-coupled miRNA mimics were transfected into AC16 cells for 48 h. Bio-miR-370-3p-MUT and Bio-NC were applied as the mutant and negative controls for the Bio-miR-370-3p group. Then, cell incubation with MagnaBind™ streptavidin magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) was conducted at 4°C overnight. The circTRRAP enrichment was detected using RT-qPCR after RNA isolation and reverse transcription.    Cardiovascular Therapeutics parallels. The mean ± standard deviation (SD) was used to indicate the final data, and statistical analysis was conducted through SPSS 22.0 (SPSS Inc., Chicago, IL, USA). The significant difference (P < 0:05) was determined using Student's t-test and analysis of variance (ANOVA) followed by Tukey's test.

Hypoxia
Induced the Significant Upregulation of circTRRAP in AC16 Cells. AC16 cells were exposed to hypoxia condition for 0 h, 12 h, 24 h, and 48 h. HIF1α protein expression was much higher in hypoxia treatment groups than that in the control 0 h group, validating the hypoxia induction (Figure 1(a)). CCK-8 results in Figure 1(b) showed that cell viability was reduced after exposure to hypoxia relative to the 0 h group. Interestingly, the relative level of circTRRAP was elevated by hypoxia in a time-dependent way (Figure 1(c)). No conspicuous change of circTRRAP expression was detected in the RNase R-and RNase R+ groups, while linear TRRAP level has been largely downregulated by RNase R treatment (Figure 1(d)). Thus, cir-cTRRAP exhibited higher stability than linear TRRAP in AC16 cells. Additionally, circTRRAP and GAPDH were enriched in the cytoplasm, but U6 was mainly determined in the nucleus of AC cells (Figure 1(e)). The result identified the cytoplasmic localization of circTRRAP in AC16 cells. High expression of circTRRAP was confirmed in hypoxic AC16 cells.

CircTRRAP
Knockdown Impeded the Damages Induced by Hypoxia in AC16 Cells. Hypoxia-induced upregulation of circTRRAP was attenuated by si-circTRRAP, which demonstrated that the transfection efficiency of si-circTRRAP was obvious (Figure 2(a)). The inhibitory effects of hypoxia on cell viability (Figure 2(b)) and proliferation (Figure 2(c)) 6 Cardiovascular Therapeutics were counteracted after cotreatment of hypoxia and si-circTRRAP. The apoptotic rate by flow cytometry manifested that knockdown of circTRRAP partly eliminated the hypoxiainduced apoptotic injury (Figure 2(d)). Furthermore, the apoptosis-related proteins were determined using western blot. The results showed that antiapoptotic Bcl-2 was upregulated while proapoptotic Cleaved caspase 3 and Bax were downregulated in the hypoxia+si-circTRRAP group contrasted to the hypoxia+si-NC group (Figure 2(e)). Downregulation of circTRRAP also abolished the promoting regulation of caspase-3 activity by hypoxia in AC16 cells (Figure 2(f)).

CircTRRAP/miR-370-3p
Signal Axis Regulated the Hypoxia-Stimulated Cell Injury. The regulatory relationship between circTRRAP and miR-370-3p was investigated in hypoxic AC16 cells. The experimental results revealed that miR-370-3p inhibition counterbalanced the stimulative influences of si-circTRRAP on cell viability (Figure 4(a)) and proliferation (Figure 4

PAWR Expression Was Upregulated by circTRRAP via
Targeting miR-370-3p. The complementary sites between PAWR 3′UTR and miR-370-3p sequences were exhibited in microT CDS ( Figure 5(a)). Then, dual-luciferase reporter assay demonstrated that miR-370-3p could negatively affect the luciferase activity of PAWR 3′UTR WT plasmid but not the PAWR 3′UTR MUT group ( Figure 5(b)). The protein level of PAWR was higher in the hypoxia group than that in the normoxia group of AC16 cells ( Figure 5(c)). The upregulation of miR-370-3p inhibited the PAWR protein expression, but this inhibition was mitigated through the transfection of PAWR ( Figure 5(d)). Moreover, western blot suggested that miR-370-3p inhibitor significantly lightened the si-circTRRAP-induced expression downregulation of PAWR ( Figure 5(e)). Collectively, circTRRAP could act as a sponge for miR-370-3p to affect the expression level of PAWR.
3.6. Overexpression of miR-370-3p Protected AC16 Cells against the Hypoxia-Induced Injury by Inhibiting PAWR Level. Furthermore, whether the function of miR-370-3p was related to PAWR was researched in hypoxic AC16 cells.

Discussion
circRNAs are characterized by covalently closed-loop structures that make circRNAs more stable to resist against RNA exonuclease than linear transcripts [16]. Our analysis indicated that circTRRAP was resistant to RNase R, and it was localized in the cytoplasm of cardiomyocytes. circRNAs have been implicated in the pathogenesis of various cardiovascular diseases, including AMI [17]. This research unraveled that circTRRAP promoted the hypoxia-induced cardiomyocyte dysfunction through the miR-370-3p-mediated PAWR upregulation. CircRNA_000203 has aggravated the development of cardiac hypertrophy through elevating the level of Gata4 via inhibiting miR-26b-5p and miR-140-3p [18]. Cir-cRNA_010567 exacerbated the myocardial fibrosis by sponging miR-141 leading to the downregulation of TGF-β1 [19]. CircMFACR modulated cell apoptosis in the heart through directly targeting miR-652-3p and indirectly upregulating MTP18 [20]. The functional experiments in this study revealed that silence of circTRRAP has resulted in the promotion of proliferation after cardiomyocytes were exposed to hypoxia. Apoptosis and inflammation are pivotal pathological aspects of AMI process [7]. The detection of apoptosis rate, apoptotic proteins, and caspase-3 activity demonstrated that circTRRAP knockdown suppressed cell apoptosis. The examination of inflammatory cytokines and oxidative indicators exhibited that the inflammatory response and oxidative stress induced by hypoxia were impeded when circTRRAP level was reduced. Significantly, the downregulation of circTRRAP could protect against the hypoxia-induced cell damages in vitro. It remains further investigation about the regulatory mechanism of circTRRAP.
Herein, we focused on the circRNA/miRNA/mRNA axis behind the function of circTRRAP in hypoxic cardiomyocytes. CircTRRAP was found to exert the sponge impact on miR-370-3p, and circTRRAP induced the negative change of miR-370-3p level. Moreover, miR-370-3p inhibitor has returned the si-circTRRAP-mediated protection for hypoxia-treated cardiomyocytes. Circ_0003204 was identified to inhibit endothelial cell injury in atherosclerosis by sponging miR-370-3p [21]. CircRNA_NEK6 retarded cell growth and invasion in thyroid cancer via the repression of miR-370-3p [22]. Our data suggested that circTRRAP could aggravate the hypoxia-induced dysfunction in cardiomyocytes by playing as a miR-370-3p sponge.
In addition, we validated that there was the target interaction between the 3 ′ UTR of PAWR and miR-370-3p. Overexpression of miR-370-3p incurred the direct downregulation of PAWR expression, and circTRRAP exerted the positive effect on the level of PAWR through mediating the miR-370-3p expression. The correlation of PAWR and circRNA was few in human research. Song et al. asserted that circMTO1 reduced gastric cancer progression via sponging miR-199a-3p to increase the level of PAWR [23]. The regulatory function of circ_0068655 in AMI was associated with the miR-498-mediated expression promotion of PAWR [14]. What is more, miR-370-3p upregulation lightened the influences of hypoxia on cardiomyocytes by downregulating PAWR. The current evidence has provided the support for circTRRAP/miR-370-3p/ PAWR axis participated in the hypoxia-induced damages of cardiomyocytes in vitro.

Conclusion
This study indicated that hypoxia treatment upregulated the expression of circTRRAP to promote the PAWR level via inhibiting miR-370-3p, then leading to the apoptotic, inflammatory, and oxidative damages in cardiomyocytes (Figure 7). CircTRRAP upregulation exacerbated cardiac injury, and circTRRAP inhibition could defend against cell dysfunction. CircTRRAP has the potential to act as a biomarker for AMI.

Data Availability
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Conflicts of Interest
The authors declare that they have no conflicts of interest.